1 “Insulin-Producing Cells ... A Preliminary Study”Cells ... A Preliminary Study” 1.Study was published in Cell Transplantation in 2018 by Ghoneim and team in Egypt. 2. “Human bone marrow–derived mesenchymal stem cells/kg” were used to form insulin- producing cells in 7 diabetes-induced mongrel dogs. 3. One dog died of pneumonia, four achieved normal sugar levels, and two dogs’ sugar levels simply reduced. 4. Removal of the TheraCyte capsules led to resurgence of diabetes. 5. The study proved that immunoisolation can be achieved with TheraCyte capsules, which were implanted in the rectus sheath of the dogs. 2 “Functional Beta ... Metabolic Control” 1. Study was published in Stem Cell Reports in 2018 by Pipeleers and team in Belgium and the United States. 2. “Human embryonic stem cell-derived pancreatic endoderm” were used to achieve “func- tional beta cell mass” (FBM) in mice. 3. As hyperglycemia was reduced, this method could help patients of diabetes. 4. Maturation of the beta cells led to their functioning similar to human pancreatic cells, determined by their secretory characteristics. 5. FBM was achieved within one year. 3 “3D Printed Porous ... Cell-Based Therapies” 1. Study was published in Scientific Reports in 2018 by Alvarez and team in Spain. 2. Macroencapsulated rodent C2C12 myoblasts and BHK fibroblasts were tested in this study. 3. Conductivity measurements of PBS in the cells in a water bath were used to understand the diffusion through the macrocapsules. 4. Applications of this study could be extended to diabetes and nervous/cardiovascular system disorders, but no specific ailments were discussed in significant detail. 5. The study proved that immunoisolation can be achieved with macrocapsules. 4 “Stem Cell Therapies ... Remaining Challenges” 1. Review was published in Cell Stem Cell in 2018 by Hebrok and team in the United States. 2. This review discussed the need to find methods to generate human stem cells that produce insulin for more facile pancreas transplants in order to reach normoglycemia in Type 1 and 2 diabetes patients. 3. The “generation of beta-like cells from human pluripotent stem cells (hPSCs)” was discussed. 4. Semipermeable encapsulation devices will allow for superior immunoprotection of islet cells and diffusion to the outside environment. 5. Human and rodent pancreatic islets were discussed and contrasted (the latter contain beta cell cores and mantles of alpha, delta, and pancreatic polypeptide cells while the former’s cells are more distributed). 5 “Colony Stimulating ... non-Human Primates” 1. Study was published in Nature Materials in 2017 by Anderson and team in the United States. 2. Immune systems of rodents, namely mice, and primates, namely cynomolgus monkeys, were studied. 3. Colony Stimulating Factor-1 Receptor inhibition could reduce fibrosis but other posi- tive macrophage functions remain. 4. Alginate capsules not leading to fibrosis could advance anti-inflammatory medicine; they are currently being used to treat Type 1 diabetes. 5. Alginate was implanted in the intraperitoneal region of the test subjects. 6 “Islet Encapsulation ... Limitations” 1. Review was published in Diabetes in 2017 by Korsgren in Sweden. 2. “Human embryonic stem cells or induced pluripotent stem cells” could allow for safe transplantation upon “removal of all HLA alleles except one.” 3. This process would be more efficient for Type 1 diabetes patients as only one allele would need to be matched. 4. Dead space in and concentration gradient of, among other factors, the macroencapsu- lation devices were discussed. 5. While pig and mice are briefly mentioned, the future of human study was the main concern. 7 “Pig-to-Primate ... Present, and Future” 1. Review was published in Cell Transplantation in 2017 by Mou and team in the United States and China. 2. Multiple factors such as age and breed affected the most opportune pigs from which to transplant islet pancreatic β-cells into primates. 3. Genetic engineering of said cells to produce more insulin may reduce the number of cells needed during transplantation and “humanize” them for their use in helping Type 1 diabetes patients achieve normoglycemia. 4. Studies with mice have shown transplantation of free islets into bone marrow and renal regions as possibly superior than the less accessible pancreatic bed. 5. The addition of mesenchymal stem cells and Sertoli cells may reduce the risk of rejec- tion of islets and allow for better oxygenation. 8 “Localized Tolerance ... Type 1 Diabetes” 1. PhD thesis was submitted in 2017 by Skoumal in the United States. 2. Microporous polyethylene glycol hydrogels returned diabetic mice to normogylcemia but had delayed glucose response; their release of TGF-β1 “also delayed rejection of allogeneic islets.” 3. FasL “can be used to create ‘induced’ immunoprivileged sites.” 4. Viruses could be utilized to transfer genes to damaged tissue reinforced with proteins that could successfully bind to the lentiviruses. 5. 160 to 270 microns is the believed ideal pore size for hydrogels; size can be modified with the addition of grain size salt. 9 “Pancreatic Islet ... Porous Membranes” 1. Study was published in Scientific Reports in 2017 by Stamatialis and team in the Netherlands. 2. A novel macroencapsulation device was developed comprised of polyethersulfone (PES) and polyvinyl pyrrolidone (PVP), allowing for glucose/insulin transfer, yet it was pro- tective from immune cells. 3. The islet cells were compartmentalized into microwells to prevent aggregation and maximize oxygenation, and the two membranes acted as a capsule and lid. 4. Human MIN6 insulin secreting cells mimicking pancreatic cells were used for experi- mentation, which occurred in e.g. glucose solutions, i.e. not in vivo. 5. The study stated that future goals include modification of the membrane “to accom- modate a population of smaller diameter islets, or pseudoislets created from stem cell derived de novo beta cells, thereby further increasing beta cell survival and function.” 10 “Considerations for Successful ... β-Cell Therapy” 1. Review was published in Cell Therapy in 2017 by Pagliuca and team in the United States. 2. Since self-administered insulin can lead to hypoglycemia in Type 1 diabetes patients, automated insulin injectors are under careful research. 3. There exist site selection considerations for transplanting islets and biomass consider- ations for their subsequent capsules. 4. A strategy to minimize the risk of hypoxia is the use of artificial oxygen carriers (AOC’s) with microencapsulated islets, allowing for better diffusion of oxygen across alginate membranes. 5. Pluripotent embryonic stem cells may be the future of supplying β-cells, but their rocky history foreshadows future complications. 11 “Co-encapsulation ... Function when Allografted” 1. Study was published in Scientific Reports in 2017 by Tuch and team in Australia. 2. Pericapsular fibrotic overgrowth (PFO) can compromise and even lead to the death of pancreatic islets used in therapy for Type 1 diabetes patients. 3. Rodent mesenchymal stem cells (MSC) stimulated with a “cocktail” of IFN-γ and TNF- α increased cytokine secretion and induced expression of inducible nitric oxide synthase to produce more nitric oxide. 4. While not affecting their viability, un- and stimulated MSC co-encapsulated with islets led to significantly greater glucose-stimulated insulin secretion, in vitro. 5. In live mice, stimulated MSC coencapsulated islets demonstrated greater viability, lower blood glucose levels, and better graft outcome; they also achieved normoglycemia earlier and for longer than their counterparts. 12 “Sox10+ Adult ... Microvascularization” 1. Study was published in Scientific Reports in 2017 by Li and team in the United States and China. 2. It has been thought that (myo)fibroblasts form the “extracellular matrix” that encap- sulate implants. 3. Analysis of rat tissue and chick embryo extract showed that Sox10+ adult stem cells found “in the stroma of subcutaneous loose connective tissues” “can be activated to proliferate and differentiate into fibroblasts at early stages of biomaterial implantation [and then] into myofibroblasts [promoting encapsulation/fibrosis] and SMCs [(Smooth Muscle Cells) or perivascular cells supporting microvessels] in vitro.” 4. Additionally, “FSP1 might be an intermediate stage marker during the differentiation of Sox10+ stem cells into perivascular cells or SMCs.” 5. “Thus, in vivo, in vitro and ex vivo results showed that Sox10+ adult stem cells contributed to both encapsulation and microvascularization,” which are considered to be competing processes. 13 “The Possible ... Encapsulated Products” 1. Review was published in Food Chemistry in 2017 by Wani and team in India. 2. Encapsulation devices are now available and being studied in the “micro- and nanodi- mensions in the form of powders, emulsions, beads, films, etc.” 3. Chemical/polymer considerations of these devices conduce their desired properties, e.g. biocompatibility. 4. Smaller sized capsules are generally more preferable to larger ones composed of the same active ingredients. 5. A nomenclature system following the following format could be used: Size (wall mate- rial, active ingredient) technology employed, where a fictional example containing size ranges and multiple wall materials listed in decreasing concentration order is 106-108 nm (β-cyclodextrin-pluronic polymer, curcumin) precipitation. 14 “Survival of Encapsulated ... Membrane Story” 1. Review was published in the World Journal of Transplantation in 2016 by Barkai and team in Israel and the Netherlands. 2. Semipermeable membranes of encapsulation devices should allow for diffusion of molecules with a radius less than 4 nm (e.g. transferrin) and prevent that of molecules with a radius of greater than or equal to 12 nm (e.g. IgM). However, harmful cytokines such as IL-1β could still ingress. 3. “The oxygen transfer rate (flux) from the plasma to the mitochondria is dictated by the oxygen gradient, the distance it has to cross, and the diffusion coefficients in the various tissues being crossed.” 4. Hyperoxia under a partial pressure of oxygen of 300 Torr can have a positive effect on transplanted islets, such as stem cell-derived β-cells, in reaching normoglycemia; rats and pigs were used for this study. 5. Finally, it is essential that “control over inflammation, cell apoptosis, angiogenesis, and the close environment of the transplanted cells” are accomplished to ensure function- ality and survival of the cells in diabetes patients. 15 “Hyperthyroidism Impairs ... Maturation in Mice” 1. Study was published in Diabetes in 2016 by Kieffer and team in Canada. 2. Not only are diabetes patients more prone to develop thyroid than the general popu- lation, but the condition can exacerbate their existing diabetic symptoms and “affect β-cell development and function.” 3. H1 human embryonic stem cells (hESC’s) were differentiated into pancreatic progen- itor cells and transplanted within TheraCyte vessels into groups of immunodeficient beige mice treated to develop acute hyperthyroid, acute & chronic hypothyroid, and euthyroid. 4. The chronic hypothyroid group experienced elevated blood glucose levels and lowered human insulin levels (perhaps indicative of the effect of thyroid hormone deficiency on β-cell maturation). The acute hyperthyroid group’s glucose levels were low. 5. Chronic and acute hypothyroid mice had significantly lowered human C-peptide levels in comparison with the euthyroid group. They also, along with acute hyperthyroid mice, demonstrated lower human C-peptide secretion. 16 “Inflammasome Components ... Body Responses” 1. Study was published in Scientific Reports in 2016 by Hayball and team in Australia. 2. To mimic foreign body response, PMMA beads were injected into the peritoneal cav- ity of mice; neutrophils and macrophages were both present at the site in significant amounts after 24 hours. Collagen, red blood cells, and blood vessels were also detected. 3. Mice deficient in ASC, an inflammasome mediator, showed greater levels of macrophages but lower levels of neutrophils than the wild-type mice; it was found that “inflammasome components ASC and AIM2 but not NLRP3 modulate the progression of fibrotic encapsulation of injected PMMA beads.” 4. Their results proposed that recruitment of mesenchymal stem cells led to activated differentiation and secretion of collagen II. 5. Thus, the unexpected link between the inflammasome components AIM2, ASC, and FBR was explored. 17 “β-Cell Replacement ... Ductal Cells” 1. Review was published in Therapeutic Advances in Endocrinology and Metabolism in 2016 by Lysy and team in Belgium. 2. Acinar cells have been investigated to determine their role as progenitors in differenti- ation to new β-cells. 3. Duct-lining cells enter epithelial-mesenchymal transition before differentiation. 4. “One of the key elements in driving pancreatic progenitors into endocrine lineage is the activation of NGN3+ expression.” 5. With treatment of growth factors, ductal progenitors differentiated in insulin-producing cells, replenishing β-cell mass in vivo and returning diabetic rodents to normoglycemia. 18 “Immunoisolation to Prevent ... Future Use” 1. Review was published in Experimental Biology and Medicine in 2016 by Shikanov and team in the United States. 2. Larger-diameter (1.5 mm) alginate capsules maintain normoglycemia for longer than those of smaller diameter (0.5 mm); barium cross-linked results in greater strength and affinity, while calcium cross-linked leads to greater compatibility. 3. Polyethylene glycol capsules allow for tuning of stiffness (key to determining foreign body response, where stiffer is more desirable) and the molecular weight of diffusing particles. Capsules with a “PEG-laminin core and an exterior inert PEG layer” or PEG-maleimide maintained cell viability, and PEG diacrylate has helped mice induced with streptozotocin achieve normoglycemia. 4. TheraCyte capsules have been shown to support pancreatic allografts and help diabetic mice achieve normoglycemia. 5. “Synthetic hydrogels are useful in tissue engineering applications as they have a high degree of reproducibility, tunability, and biocompatibility, whereas natural hydrogels have a higher degree of biological specificity.” Encapsulation research could potentially be extended to support transplantation of ovarian follicles to treat ovarian insufficiency. 19 “Assessment ... Macroencapsulation Device” 1. Study was published in Transplantation in 2016 by Tang and team in the United States. 2. “T-cells are the primary drivers of alloimmune responses” and are activated directly or indirectly by MHVC or peptides. The former requires proximity to transplanted cells while the latter does not necessarily. 3. Diabetic mice underwent transplants of pancreatic progenitor cells or MIN6 cells; embryotic pancreatic cells had the highest likelihood to differentiate into insulin-producing cells but could indirectly active T-cells. 4. ViaCyte, similar to TheraCyte, devices were charged with embryonic pancreata, new- born pancreata, or MIN6 cells and implanted into the mice. The capsules could prevent T-cell stimulation. 5. Capsules were also successful at protecting the β-cells from autoimmune attack, as well as the MIN6 cells from immune responses. 20 “Implanting 1.1B4 ... Deficient Mice” 1. Study was published in the World Journal of Diabetes in 2016 by Flatt and team in the United States and Ireland. 2. Human β-cell line 1.1B4 can secrete insulin and is responsive to glucose; it is “created by the electrofusion of freshly isolated human -cells with immortal PANC1 epithelial partner cells.” 3. Female mice were induced with diabetes via injection of streptozotocin, and pseuodislets or suspensions of the 1.1B4 cells were also administered to the two experimental groups. 4. Glucose levels of the mice containing suspensions and pseudoislets were lower than those of the control group; the latter group’s levels were most comparable to normal. 5. The pseudoislet clusters may still have tumorigenic properties that were not observed in vitro due to hypoxia, whereas their ease of vascularization in vivo allows proliferation. 21 “Progress and Challenges ... Bioartificial Pancreas” 1. Review was published in Nano Convergence in 2016 by Jun and team in the United States. 2. A primary concern for extravascular macrocapsules such as the TheraCyte bioartificial pancreas and its diffusion chamber is conglomeration of the islets, which could lead to hypoxia and reduced flow of nutrients—layered alginate may remedy this but with its own complications. 3. Vascular macrocapsules such as coiled/hollow tubes are more prone to fibroblast attack and have been studied in trials with diabetic dogs, humans, etc. 4. Thermoplastic membranes, inorganic membranes made of metal oxides, synthetic hy- drogels, and microcontainers also serve as currently-researched macrocapsules. Algi- nate, agarose, PEG, and peptide amphiphile are microcapsule examples. 5. Surface modification of the islets and microfluidic devices/micropatterned surfaces are alternatives to encapsulation devices that could prevent against autoimmune attack of transplanted embryonic stem cell-derived pancreatic β-cells. 22 “Cell Therapies ... Beta-Cell Replenishment” 1. Review was published in the Italian Journal of Pediatrics in 2016 by Iughetti and team in Italy. 2. A detailed explanation of the maturation and biology of stem cells, such as endodermic (from liver or pancreas), haemopoietic, and mesenchymal is explored. 3. “The transplantation of ESCs-derived pancreatic progenitors, rather than full differen- tiated ESCs-derived beta cells, proved to be more effective in restoring normoglycemia by means of the glucose-dependent release of insulin.” 4. Despite their advantages compared to adult stem cells, totipotent ESC’s face setbacks such as ethical dilemmas and expensive resources needed for research. 5. Stem cells from the placenta, such as human amniotic epithelial cells, show promise for maintaining glucose levels and averting tumors in diabetes patients, as they have done in studies with mice. 23 “Concise Review ... Type 1 Diabetes” 1. Review was published in Stem Cells Translational Medicine in 2016 by Ling and team in Belgium. 2. Using islets from the pancreas of pigs is a promising source of β-cells for diabetic patients, and studies with rodents and primates have displayed this. 3. “Human stem cell-derived grafts can form β-cell implants in immune-deficient mice.” 4. Human embryonic stem cell-derived pancreatic endoderm grafts resulted in an endocrine purity of double that of human islet grafts. 5. Further research should clarify the specific metrics associated with stem cell-generated human β-cells versus pancreatic primary human β-cells, and what biology defines a “true” mature human β-cell. 24 “Progress and Challenges ... Devices” 1. Review was published in Biotechnology and Bioengineering in 2015 by Roy and team in the United States. 2. A particular drawback of vascular perfusion devices is the potential to initiate lethal blood coagulation and thrombosis. Extravascular macrocapsules evade this problem but face the disadvantage of requiring large volumes in small bodily spaces. 3. Hollow fibers, bag-like structures, polymeric hydrogel sheets, and planar membranes are examples of macrocapsule morphologies. 4. The review features a table including information about allo- and xenogeneic islet transplants, their results, number of islets, etc. 5. Nano-sized pores can be utilized to block passage of large immune molecules into islet capsules, but they must be refined to selectively allow diffusion of necessary smaller molecules. 25 “Long-Term ... Competent Mice” 1. Study was published in Nature Medicine in 2016 by Anderson and team in the United States. 2. Human pluripotent stem cells (hPSCs) that differentiate in vitro into pancreatic β-cells expand the supply of insulin-producing tissue. 3. “TMTD alginate–encapsulated SC-β cells provide long-term glycemic correction and glucose responsiveness without immunosuppressive therapy in immune-competent diabetic C57BL/6J mice” induced with streptozotocin, as these specific rodents “pro- duce a strong fibrotic response and FBR similar to that observed in human patients.” 4. 1.5-mm TMTD alginate spheres were the most successful at achieving normoglycemia, yet significantly different results were found when assessing rat versus human cell islets. 5. “Maintenance of insulin-positive cells after retrieval of the implant suggests that the SC-β cells retain their differentiation state over the course of the entire study.” 26 “Quantitative ... Generation Imaging” 1. Study was published in PLOS One in 2015 by Bratlie and team in the United States. 2. Over the course of foreign body response, fibroblasts and their derivatives, myofibrob- lasts, secrete types I and III collagen to form a barrier around the implant, rendering its functions obsolete. 3. Type III collagen is of the minority in healthy tissue, but its role in FBR is crucial as a deficiency can lead to the formation of scars, where its orientation is in parallel bundles unlike the isotropic orientation in healthy tissue. 4. Second harmonic generation microscopy was used for its ability to measure collagen alignment. 5. Polystyrene beads were injected into female mice to induce FBR, and the orienta- tion of collagen in the fibrotic overgrowth was analyzed to demonstrate that surface modifications can be made to implants to encourage the desired collagen production outcome. 27 “Pancreatic Tissue ... Immune-Mediated Diabetes” 1. Study was published in Cell Transplantation in 2016 by von Herrath and team in the United States and Germany. 2. Immunosuppressants taken to avert rejection of transplants can put patients at risk of cancer and other diseases; they can also be toxic to insulin-producing β-cells. 3. Mice were injected with lymphocytic choriomeningitis virus (LCMV), leading to a cytotoxic T-lymphocyte response, mimicking the effects of Type 1 diabetes. 4. The TheraCyte capsules were charged with neonatal pancreatic tissue due to its ability to better withstand the effects of hypoxia than adult tissue. 5. The capsules maintained euglycemia in the mice infected with the virus and protected the cells from attack, along with creating an environment in which the cells could survive for long periods of time. 28 “Innate Immunity ... Friends or Foes” 1. Review was published in BioMed Research International in 2015 by Hayball and team in Australia. 2. Foreign body response begins with protein adsorption, after which neutrophils are recruited to induce inflammation. Macrophages and fibroblasts work to destroy and surround the implant, reducing its function. 3. “Alarmins,” integrin receptors, toll-like receptors, inflammasomes, and more also take part in the process of identification of pathogens and recruitment of cytokines. 4. Surface chemistry and surface topography, which analyze functional groups that could contribute/take away from protein adhesion and the texture of the membrane that could be most compatible, are the key considerations to reduce FBR in implants. 5. Continued research using mice “deficient in key innate immune effector response path- ways” is necessary to understand the effects of surface chemistry alterations on in vivo FBR. 29 “Bioengineered ... Immune-Mediated Diabetes” 1. Review was published in the World Journal of Transplantation in 2015 by Shapiro and team in Canada. 2. The “Edmonton Protocol” was established after the first example and precedent for islet transplantation for Type 1 diabetics, who were insulin-independent for a year before they again required infusions. 3. Pancreatic kidney transplantation and islet transplantation have both been conducted in human and animal models and have been able to achieve relatively long-term insulin independence. 4. Porcine xenografts and human endoderm stem cells are the current focus due to advan- tages over allogeneic transplantation, including a minimized need for immunosuppres- sion, practically unlimited supply, and resiliency of the cells themselves. Differentiating β-cells from inducible pluripotent stem cells could minimize the teratoma risk. 5. The criteria for a successful transplantation site includes volume of site/transplanted tissue, blood and oxygen supply, “access to physiological blood glucose levels,” “ease of access and the potential for rapid retrieval,” and “minimal early inflammatory reaction and promotion of long-term survival.” 30 “Polycaprolactone ... Cell-Encapsulation Devices” 1. Study was published in ACS Nano in 2015 by Desai and team in the United States. 2. Polycaprolactone (PCL) thin-film macrocapsules were used in this study to reap the benefits of permeability, flexibility, and membrane control. 3. The capsules fabricated with zinc oxide and polyethylene glycol contained nanoscaled features that allowed for fine-tuning of the capsules’ membranes, and the careful timing of decomposition of the material could remove the necessity for retrieval when used in diabetes patients. 4. The β-cells encapsulated in the nanodevices maintained glucose levels without perme- ation of toxic external cytokines. 5. A steady rate of increasing vascularization was observed around the capsules in vivo in mice after two weeks. 31 “Development ... Therapy for Diabetes” 1. Review was published in Expert Opinion on Biological Therapy in 2013 by Ricordi and team in Italy and the United States. 2. In macrocapsules, the device thickness should be minimized to allow for facile diffusion; device thickness and islet density have an inverse relationship. Microcapsule geometry is more advantageous with regards to diffusion and foreign body response. 3. “Capsule biocompatibility depends on the capsule chemical composition, surface charge, porosity, surface roughness, implant site and shape.” 4. “Among the sites that have been explored are the skin, the pancreas, the submandibu- lar gland, the muscle, the omentum, the bone marrow, the gastric submucosa, the gen- itourinary tract, the kidney capsule, the anterior eye chamber, the testis, the spleen, the brain, and the thymus;” alternative sites include the omental pouch and gastric submucosal space for implantation. 5. The expert opinion of the reviewers is that an on-site oxygen supply and anti-inflammatory agent delivery are crucial to islet survival and function for diabetes patients. 32 “Minireview: Directed ... Future Considerations” 1. Review was published in Molecular Endocrinology in 2015 by Hunter and team in the United States. 2. In vitro differentiation of stem cells has produced β-like cells that differ genetically from the desired β-cells for transplantation, and the question of whether or not these cells are fully mature and functional remains. 3. The review suggests that “perhaps implantation of progenitor cells is indeed a better choice for generating complementary islet cell types.” 4. TheraCyte-encapsulated cells have evidence of being resistant to immune attack upon transplantation, and the subject diabetic mice remained normoglycemic as the cells successfully produced insulin. 5. A layer-by-layer technology of encapsulation devices may allow for careful control over diffusion, immune response, and biocompatibility. 33 “Size- and Shape-Dependant ... Primates” 1. Study was published in Nature Materials in 2015 by Anderson and team in the United States and Slovakia. 2. Past research has found that circular-shaped, thin, and large pore-bearing implants lead to the least aggressive FBR. Smooth surfaces without acute angles are more bio- compatible. 3. Barium cation-cross-linked spherical alginate hydrogel spheres were embedded in the intraperitoneal space of mice. 4. FBR was reduced as sphere size, not total surface area, increased. This trend continued with other smooth materials and in larger rodents as well. 5. STZ-induced diabetic mice and primates were implanted with the 1.5 mm capsules containing rat islets, and the larger spherical capsules were able to “ward off cellular deposition for at least 6 months.” 34 “Treatment of Diabetes ... Current Developments” 1. Review was published in the Journal of Zhejiang University Science B in 2015 by Wang and team in China. 2. Neonatal pig islets are more likely to be successful in transplants over aged ones; they then differentiate into mature β-cells. 3. Genetically modifying pigs providing islets could make the donated islets less prone to inflammation or premature death. 4. In 2008, a study by Prochorov and team implanted intravascular nylon microporous macroencapsulated fetal rabbit islets in Type 1 diabetes patients without immunosup- pression. At follow-up two years later, insulin demands were reduced, and C-peptide was raised without detrimental side effects. 5. The review features a studies table including information about islets used, implanta- tion site, encapsulation material, advantages, and limitations. 35 “Human Embryonic ... Cell Escape” 1. Study was published in Stem Cell Research in 2014 by Ansari and team in the United States. 2. One of the risks associated with using human embryonic stem cell-derived cells is the accumulation of teratomas. 3. TheraCyte capsules were implanted in mice, and bioluminescent imaging was utilized for data collection. 4. Maturation of the stem cells into islets occurred without a significant increase in biomass/growth. 5. No tumors were found in the implantation sites, and the capsules maintained normo- glycemia despite alloxan-induced diabetes. 36 “Islet and Stem ... Clinical Transplantation” 1. Review was published in The Review of Diabetic Studies in 2013 by Lakey and team in the United States. 2. The International Pancreas Transplantation Registry, International Pancreas and Islet Transplantation Association, and European Study Group in simultaneous Pancreas and Kidney Transplantation are global organizations dedicated to the support of research for transplants for diabetics. 3. “Between 2007 and 2010, islet graft survival at 1 year (92%) and 3 years (83%) is comparable, if not slightly superior, to survival rates demonstrated with whole pancreas transplants (80% and 61% at 1 year and 3 years, respectively).” 4. Prevascularized devices are among the several options for implantation device designs, and they encourage contact with the surrounding blood vessel network. 5. It was found that there is no evidence of true insulin independence being achieved using stem cell-derived β-cells. 37 “Composition and Function ... Cell Grafts” 1. Study was published in the American Journal of Physiology-Endocrinology and Metabolism in 2014 by Pipeleers and team in the United States and Belgium. 2. Human embryonic stem cell-derived pancreatic endoderm were implanted in nondiabetic mice either alone, microencapsulated in alginate, or macroencapsulated in TheraCyte devices. 3. Mice implanted with human islet cell graft acted as the control group. 4. “Implants in a macrodevice thus exhibited a higher endocrine and cell purity than the average human islet cell graft prepared from donor pancreases.” 5. Free and macroencapsulated islets exhibited vascularization while microencapsulated ones did not. 38 “Current Status of Islet Encapsulation” 1. Review was published in Cell Transplantation in 2014 by Lakey and team in the United States. 2. Thus far, alginate has received the most focus as an encapsulation material as it is “stable to oxidative damage in vivo.” 3. The Soon-Shiong, Basta, Califiore, Tuch, and Valdes-Gonzalez groups and more have conducted clinical trials on humans in small quantities. 4. As a result of a lack of donor tissue, xenotransplantation and stem cells are methods being explored to provide diabetics with the insulin sources they need. 5. The review features a comprehensive table spanning several pages including informa- tion about the islet source, animal recipient, capsule material, outcome, and authors of nearly 100 studies on the subject. 39 “Encapsulated Islets ... Requiring Solution” 1. Review was published in Advanced Drug Delivery Reviews in 2013 by Scharp and team in Italy and the United States. 2. The major companies that have been involved in macrocapsule research are ViaCyte, Betalogics, Islet Sheet Medical, and Beta-O2; those involved in microcapsule research are Living Cell Technologies, Islet Sciences, EncapsuLife, DefyMed, and Prodo Labo- ratories, almost all of them for less than the last five years. 3. While experimentation has been conducted using pig, dog, rodent, and primate islets, clinical trials on humans are still in the future. 4. Some of the greatest concerns regarding expanding encapsulation research to human clinical trials include non- and immuno-responses to the cells, loss of islet mass, the use of immunosuppression, donor sensitization, and lack of resources. 5. Current research is being done on coating islets in biocompatible living cells, such as a lipid-PEG-biotin and streptavidin layer, to prevent an immune response. 40 “Macroecapsulation of Pancreatic ... Therapy?” 1. Review was published in the Journal of Clinical Developmental Biology in 2015 by Polidori in the United States. 2. Human embryonic stem cells differentiate to produce insulin-secreting pancreatic en- doderm, and they can withstand cryopreservation and exhibit high immune tolerance. 3. Encaptra by ViaCyte and TheraCyte can prevent cell escape with their semipermeable membranes/bilaminar properties. 4. “Progenitors may contribute to the tumorigenic potential of the transplanted cells.” 5. The next steps include addressing the size requirement (possibly 46 meters) of the planar devices and their permeability to immune system elements that cause fibrosis and necrosis. 41 “β-Cell Regeneration ... ‘Holy Grail’?” 1. Review was published in the Journal of Molecular Endocrinology in 2014 by Stanley and team in Australia. 2. Loss of consciousness and inability to be awoken are examples of symptoms diabetics afflicted with hypoglycemia are unaware they have; diabetic neuropathy is also a com- plication. 3. Two types of immortal pluripotent stem cells exist: embryonic stem cells and induced PSCs, “derived by reprogramming somatic cells to an ESC-like state.” Im- munosuppression is required for β-cells derived from either cell when transplanted. 4. The induction of definitive endoderm using activin A, the employment of retinoic acid on definitive endoderm to induce foregut and derive pancreatic endoderm, and the gen- eration of endocrine/exocrine cells after use of noggin, dorsomorphin, or dorsomorphin homolog 1 are the key steps in differentiation to pancreatic progenitors. 5. Improving differentiation efficiency, purifying cell clusters, micro- and macroencapsu- lation methods (e.g. TheraCyte capsules), and removing unwanted cells are ways by which immunorejection/teratoma formation can be avoided. 42 “Enrichment of Human ... In Vivo” 1. Study was published in Stem Cells in 2013 by Kieffer and team in Canada. 2. Pancreatic progenitor cells (second stage of pancreas development) and polyhormonal cells differ by their expression of NKX6.1. 3. Mice were induced with diabetes and subsequently implanted with TheraCyte capsules loaded with human embryonic stem cells. 4. “Progenitor cells enriched for NKX6.1-positive cells matured more quickly in vivo into glucose-responsive insulin-secreting cells compared to an endocrine-enriched progenitor population.” 5. NKX6.1-enriched cells exhibited indicators of mature pancreatic β-cells, one being “robust nuclear MAFA expression.” The line enriched for polyhormonal endocrine cells “developed into mostly mature α-cells following transplant and produced immature pancreatic β-cells, that did not secrete insulin in a glucose-responsive manner or express nuclear MAFA.” 43 “Review: Macro-Encapsulation ... Hydrogel” 1. Study was published in the Journal of Medical and Biological Engineering in 2013 by Inoue and team in Japan, China, and Taiwan. 2. “Serious complications such as chronic renal failure, retinopathy, and neuropathy fre- quently progress in severe-DM patients, in whom β-cell function is virtually lost.” 3. Rat islets were encapsulated in polyvinyl alcohol hydrogel and secreted insulin for longer in vitro than free islets. 4. Diabetic mice returned to near-normoglycemia as a result of implantation and gained weight; the encapsulated islets could be cryopreserved without losing function; fibrosis surrounding the capsules was found but penetration into the islet region was not. 5. “TheraCyte is unsuitable for clinical diabetes therapy because of its limited volumetric capacity (largest capacity: 40 µl).” 44 “The TheraCyte ... Immunized Hosts” 1. Study was published in Cell Transplantation in 2013 by Tibell and team in Sweden. 2. Rats were immunized via transplantation of other rat islets—antidonor antibodies were produced. Splenocytes were also used for immunization. 3. Rats were implanted with empty TheraCyte capsules, which were then filled with islets some days after the rats were induced with diabetes. 4. Even though the pore size of TheraCyte capsules would in theory allow passage of antibodies, inflammation around the deices and harm to the islets within were not discovered, and the islets helped reach normoglycemia; this is significant because many diabetes patients are immunized from previous transplants. 5. The “study did not suggest that encapsulated islets enhanced [CD8+] T-cell activity over time.” 45 “Inconsistent Formation ... Nude Mice” 1. Study was published in the The American Journal of Physiology: Endocrinology and Metabolism in 2010 by Butler and team in the United States. 2. Rats were implanted with TheraCyte-encapsulated or free pancreatic endoderm derived from human embryonic stem cells. 3. Endocrine development of the implants was not as promising as expected; human C- peptide/insulin secretion was also limited. 4. Exocrine duct-like tissue was more so developed, along with some islet-like tissue, versus the desired cell structure. 5. Theracyte capsules did not offer a supportive environment for the maturation of cells, and insulin secretion was not responsive to glucose levels. 46 “Real-Time Bioluminescence ... (Macaca Mulatta)” 1. Study was published in Transplantation in 2009 by Ansari and team in the United States. 2. The immune system of rodents is not as complex as that of humans, but “there are important similarities between human and nonhuman primate MHC genes and devel- opment of the T-cell repertoire.” 3. Rhesus monkey skin fibroblasts treated to express luciferase were implanted in rhesus monkeys inside TheraCyte capsules. 4. The capsules in this study provided sufficient immunoprotection, and the graft survived for an extended period of time, as shown by in vivo bioluminescent imaging. 5. Photon count decreased one week after implantation, but this could have been caused by either cell death or lack of expression of the luciferase gene—the latter was especially likely as the fibroblasts were at the log phase of growth. 47 “Human β-Cell ... Cell Therapies” 1. Study was published in Transplantation in 2009 by Ansari and team in the United States. 2. Mice induced with diabetes were implanted with TheraCyte-encapsulated murine neonatal islets or human fetal pancreas cells. 3. “Human β-cells survive, differentiate, and function within a durable immunoisolation device;” insulin secretion was responsive to glucose levels. 4. Although survival in the capsule was not ideal, antiapoptotic drugs may aid in future experiments. It was observed that cell death occurred abruptly and then recovered over time. 5. The study also found that “in transplanted NOD mice, T cells were not recruited to the tissue surrounding the device.” However, another study found contrasting results. 48 “Treatment of Diabetic ... Encapsulated Islets” 1. Study was published in the Journal of Cellular and Molecular Medicine in 2008 by Osborne and team in the United States. 2. BB rats and streptozotocin-injected rats were used to mimic the effects of diabetes, which was onset prior to implantation, as is most often the case with humans. 3. Rat pancreatic islets were loaded into TheraCyte capsules (which are comprised of “an inner immunoisolation membrane and an outer vascularization membrane”) and implanted subcutaneously into the diseased rats. 4. Neither experimental group achieved normoglycemia; STZ-induced diabetes was shown to be less severe than that in the BB rats, and glucose levels were lowered more dramatically in the former. 5. The islets did help to control weight loss in both groups after several weeks. 49 “Preimplantation ... Rodent Model” 1. Study was published in Transplantation in 2008 by Tibell and team in Sweden. 2. TheraCyte capsules get “well vascularized in rodents and humans, and [...] allografts can survive up to 1 year inside.” 3. Female mice were implanted with empty capsules, then induced with diabetes, and then injected with male rat islets. 4. Mice with preimplanted chambers showed higher cure rates, higher body weight gain, greater ability to lower blood glucose, and higher volume densities of endocrine tissue. 5. Fewer islets were necessary in preimplanted capsules, achieving the quantity used when freely inserted into the kidney capsules. 50 “Treatment of Osteoporosis ... Rat Model” 1. Study was published in Osteoporosis International in 2006 by Lu and team in Taiwan. 2. Transplantation of parathyroid tissues presents an alternative to daily injection of parathyroid hormone (PTH) to treat osteoporosis. 3. Human parathyroid tissue was loaded into TheraCyte capsules and implanted into overiectomized rats. 4. Body weight of the rats increased after four months, and no bone tumors were detected after seven months. 5. The cells secreted iPTH, and FBR did not occur; bone mineral density increased as a result of the encapsulated cells. 51 “Soluble Factor(s) ... Stem Cells” 1. Study was published in Experimental Hematology in 2005 by Anderson and team in the United States. 2. Mice received empty TheraCyte capsules, which were charged with bone marrow Lin- cells after the animals underwent lethal radiation exposure. 3. “3T3, stromal cells, CHO, 3T3-L1, endothelial cells, [and] whole bone marrow cells” did not provide the curative effect the Lin- cells did for the mice. 4. The TheraCyte capsules provided an environment where the cells could differentiate into myeloid and lymphoid cells. 5. Since donor cells did not leak from the capsules, soluble factors ultimately determined the rescuing effect. 52 “Xenotransplantation ... Liver Cells” 1. Study was published in Transplantation Proceedings in 2005 by Elliott and team in the United States and New Zealand. 2. “Porcine hepatocytes [...] are abundant, maintain their differentiated functions and metabolic activity, and do not present the tumorigenicity and de-differentiation risk of modified cells.” 3. Porcine liver cells were transplanted into mice in TheraCyte capsules and maintained viability until extraction. 4. The cells released albumin and factor 8 increasingly in culture and retained viability for weeks; they also uptook and released indocyanin green. 5. Although it has so far proved difficult to accomplish, “maintaining and proliferating neonatal hepatocytes [in vitro] has many practical advantages” for treating hepatic diseases. 53 “Transplantation ... Mice and Monkeys” 1. Study was published in Transplantation Proceedings in 2005 by Bambra and team in the United States, New Zealand, Singapore, and Italy. 2. Porcine islets were transplanted into diabetic mice and nondiabetic monkeys in alginate or TheraCyte capsules. 3. 60% of mice with microencapsulated islets reached normoglycemia without immune reaction. Mice with macroencapsulated islets reached normoglycemia for several weeks. 4. Insulin dependence resurged when the implants were removed. 5. The alginate capsules proved to be less permeable than the TheraCyte capsules, al- lowing for greater control over rejection. 54 “Cell-Based ... Autoimmune Encephalomyelitis” 1. Study was published in Gene Therapy in 2005 by McMillan and team in the United States. 2. Current belief holds that CD4 T-cells are activated and secrete inflammatory cytokines to initiate specific destruction of the myelin sheath of axons, leading to multiple sclerosis. 3. Mice were injected with pertussis and myelin basic protein (MBP) to induce murine experimental autoimmune encephalomyelitis (EAE), and proteolipid protein (PLP)- secreting and MLB-secreting fibroblasts treated the disease. The specific fibroblast line used for treatment was immaterial. 4. Replacing the signal sequence to one “from the secreted protein rat KC chemokine” reduced the dosage of cells needed for therapy. 5. Sequestering the PLP-secreting cells in a chamber allowed for allogeneic tissue to be used, and the chamber itself, although inducing a local response, had no effect on the disease. 55 “Porcine Endogenous ... Islet Transplant” 1. Study was published in the American Journal of Transplantation in 2004 by Korbutt and team in Canada. 2. Porcine endogenous retrovirus (PERV) shares characteristics with the mouse and feline leukemia viruses, which cause leukemia in those infected. “If PERV incorporates into a human germline, it will be passed to subsequent generations, allowing potential rescue of the virus by later recombination events with other endogenous retroviruses.” 3. Porcine pancreas islets were freely or alginate-microencapsulated and implanted in mice induced with diabetes; others were TheraCyte-macroencapsulated and implanted with splenocytes in immunodeficient mice. 4. PERV DNA was detected in the mice at the site of transplant as well as the liver and spleen, and the PERV polymer gene was expressed by the tissues, regardless of microencapsulation. PERV was not detected in tissue encapsulated by TheraCyte. 5. There was a correlation between destruction of porcine tissue and PERV, suggesting that a lack of PERV detection was a result of rejection of graft tissue. 56 “Long-Term Erythropoietin ... Bioisolator Devices” 1. Study was published in Human Gene Therapy in 2003 by Osborne and team in the United States. 2. “Current indications for the use of [erythropoietin (regulates erythrocyte production and ‘stimulates the maturation and increases the survival of erythroid precursor cells located principally in the bone marrow’)] include treatment of anemia caused by renal insufficiency, cancer or chemotherapy for cancer, and anemia of prematurity,” as well as reducing blood transfusions. 3. Rats were implanted with autologous and allogeneic transduced smooth muscle cells encapsulated in TheraCyte capsules, and elevated hematocrit levels indicated that both implants sustained erythropoietin expression. 4. Removal and reimplantation of capsules did not lead to an immune response, and hematocrit levels rose quickly after. Devices overall showed no fibrosis and good vas- cularization. 5. It is estimated that a human would require about 5 40-microliter TheraCyte devices, which could help patients in need of erythropoietin or insulin for diabetes. 57 “Retroviral Packaging ... Gene Delivery” 1. Study was published in Frontiers in Bioscience in 2003 by Dornburg and team in the United States. 2. TheraCyte capsules were loaded with “retroviral vector producer cells, which release vectors derived from spleen necrosis virus, [(SNV) ...] an avian reticuloendotheliosis virus.” 3. “D17 is a canine osteosarcoma cell line. DSH-cxl7A5 [in the capsules] is a[n] SNV derived retroviral vector packaging cell line that was derived from D17 cells.” 4. In vivo analysis taking place in immunodeficient mice given D17 to develop tumors showed that the retroviral vectors were indeed released from the non-radiated cells in the capsules, as the tumors expressed bacterial beta-galactosidase gene regardless of the site of implantation. 5. In vitro analysis showed that the vectors were released for half the time in radiated cells than non-radiated ones. 58 “Survival of Pancreatic ... TheraCyte Device” 1. Study was published in Transplantation Proceedings in 2002 by Nadler and team in the United States. 2. Rat pancreatic islets were injected into female diabetic mice. 3. Use of TheraCyte capsules extended longevity of the islets and led to euglycemia; vascularization around the capsules was detected. 4. After just a couple of days post-removal of implants, the rats returned to being hyper- glycemic. 5. “The NOD model is considered the best animal model for studying human autoim- mune diabetes” due to the similarity between humans and female mice to “develop spontaneous insulitis.” 59 “Survival of Macroencapsulated ... Humans” 1. Study was published in Cell Transplantation in 2001 by Wernerson and team in the United States and Sweden. 2. Those suffering from chronic hypoparathyroidism after (para)thyroid surgery can have hyper- and hypocalcemic episodes due to unstable calcium levels. 3. Human parathyroid tissue was loaded into TheraCyte and Boggs membranes for macroencapsulation and implanted in human patients, along with their own parathy- roid tissue in Boggs devices, without immunosuppression. Implantation in mice acted as controls. 4. Fibrotic overgrowth was detected for all of the allogeneic implants; the results for the other implants with mice controls matched well with the result of the mice. 5. “Parathyroid tissue can survive for at least 4 weeks when encapsulated in this bilayered membrane and implanted SC in humans.” 60 “Baboon Mesenchymal ... In Vivo” 1. Study was published in Human Gene Therapy in 2001 by Devine and team in the United States. 2. Human mesenchymal stem cells (hMSC’s) quickly multiply in culture, and transduced stromal cells are long-lived and thus able to secrete a transgene product for an extended period of time. 3. In vitro hEPO-transduced baboon and human MSC’s were implanted in TheraCyte capsules in diabetic and immunodeficient mice and baboons, which were similar in their expression of hEPO and elevation of hematocrit. 4. Overall immune response was mild, and factors that are speculated to have contributed to the loss of expression of hEPO over time include gene silencing, promoter inactiva- tion, and impaired gene regulation. 5. This study provided the proof-of-concept that “MSCs could be genetically modified to secrete a therapeutic protein.” 61 “Protection of Xenografts ... Anti-CD4 Antibody” 1. Study was published in Cell Transplantation in 2001 by Lew and team in Australia. 2. Immunodeficient mice were implanted with TheraCyte-encapsulated pig kidney (PK) cells, hamster ovarian cells, and a mouse insulinoma cell line. 3. Empty capsules elicited a nonspecific immune fibrotic response, and vascularization was also detected. Loaded capsules kept cells alive both in vitro and in vivo. 4. Encapsulated PK cells did not survive in nonimmunodeficient mice, but did so when they were treated with anti-CD4 antibodies. Unencapsulated PK did not survive in the latter case. 5. Lymphocytes/CD4+veT cells play the most significant role in the immune response, and they are called to the device surface via antigen secretion. Nitric oxide and cy- tokines from the phagocytes can harm the grafts. 62 “Improved Vascularization ... Growth Factor” 1. Study was published in Cell Transplantation in 2000 by Weir and team in the United States. 2. β-cells consume more oxygen as glucose levels increase, so in a hyperglycemia diabetic environment, oxygen distribution in an islet transplant can be severely insufficient. β-cells have overall a higher oxygen requirement than other cell types. 3. Vascular endothelial growth factor (VEGF), which promotes vascularization, was in- fused into TheraCyte capsules, which were implanted in rats. 4. High-dose VEGF transfusion led to significantly larger and greater quantities of blood vessels in comparison to low- and no-dosage, and insulin diffusion was seemingly more efficient as a result. 5. “Integrins, transforming growth factor-P, platelet-derived growth factor, angiopoitin-1, and other factors may all be required to form fully functional vessels.” 63 “Longitudinal Studies ... Doppler Technique” 1. Study was published in Cell Transplantation in 2000 by Tibell and team in Sweden. 2. A previous study found that glucose exchange at day 1 and after 3 months of implan- tation of TheraCyte devices were similar, while that at 4 weeks was reduced; however, vascular count was not consistent with these results (they increased from day 1 to 4 weeks). 3. A laser Doppler probe (helium-neon laser light, 780 nm, traveling through a fiber optic cable) was inserted into TheraCyte devices through the polyethylene port typically utilized for islet loading. 4. The devices were implanted in rats and it was found, via signal analysis, that perfusion of the surrounding tissue reduced at 4 weeks and recovered after 3 months. 5. Inflammation occurs just following implantation. Neovascularization proceeds this pro- cess, which increases in prevalence until a vascularized fibrotic layer develops at about 4 weeks. Full perfusion is achieved after several weeks. 64 “In Vivo ... Microdialysis Technique” 1. Report was published in Cell Transplantation in 1999 by Tibell and team in Sweden. 2. The diffusion barrier of capsules could delay glucose response and initiation/completion of insulin secretion into circulation, leading to hyper- and hypoglycemia respectively in diabetes patients, compared to the faster reaction time of the natural human pancreas. 3. The microdialysis probe has two ports and a semipermeable membrane at the apex. Substances in the extracellular fluid diffuse into the continuously pumped perfusion medium and are collected. Exogenously infused compounds like glucose can also be studied. 4. The amount of glucose recovered will not represent exactly the amount in the extracel- lular fluid and depends on the thickness/length of the probe, concentration of glucose, etc. In vitro analysis first assesses the recovery rate of the probe. 5. Glucose diffusion through TheraCyte capsules in vivo in rats was found to be delayed at first but then reach similar kinetics to subcutaneous fat. 65 “In Vivo Studies ... Microdialysis Technique” 1. Study was published in European Surgery Research in 1999 by Tibell and team in Sweden. 2. “Insulin was injected into Theracyte” devices, which were transplanted in rats following the microdialysis technique. 3. While after 1 day a change was not detected, after some weeks the insulin recovery was greatly reduced, but then consistent with the control group at three months. 4. Vascularization surrounding the implants was also more prominent at several weeks and three months than just one week post-implantation. 5. It is recommended that the capsules be charged with grafts after allowing neovascu- larization to occur. 66 “In Vivo Delivery ... Immunoisolation Devices” 1. Study was published in the Journal of Molecular Medicine in 1999 by Johnson and team in the United States. 2. Three clones of MSU1.2 human fibroblast cells “engineered to express human growth hormone [(HGH)] as an indicator gene under control of the CMV promoter,” acting as therapeutic peptide, were loaded into TheraCyte capsules and implanted in rats. 3. After removing the capsules, expression of HGH starkly reduced. 4. It was determined that cells could not have escaped the capsules and expressed HGH. 5. Advantages of TheraCyte are presented, including the fact that it keeps implanted cells in a cluster, allowing for more facile extraction. 67 “Correction ... Immunoisolation Devices” 1. Study was published in the Journal of Molecular Medicine in 1999 by Johnson and team in the United States. 2. Insulin injection is not a long-lasting cure for diabetes; rather islet transplantation can eradicate the risk of diabetic nephropathy and simultaneously prevent hypoglycemia. 3. Cyclophosphamide induced diabetes in female NOD mice, and they were implanted with insulinoma (NOD-RIP-Tag insulinoma cell line NIT-1)-charged TheraCyte cap- sules. 4. The insulinomas responded positively to glucose tolerance tests, regulating blood glu- cose levels at a normal level, without immunosuppression needed. 5. The capsules’ finite size also prevented the immortal growth of the insulinomas, which would have led to hypoglycemia. 68 “Reversal of Hyperglycemia ... Islets” 1. Study was published in Transplantation in 1999 by Weir and team in the United States. 2. Diabetes-induced mice were implanted with rat islets inside TheraCyte capsules, which were either pre-planted, suspended in culture medium, or suspended in alginate cross- linked with barium chloride. 3. Alginate-containing devices showed less vascularized fibrotic overgrowth than the other medium, but insulin release was not impacted in either case by immune response. However, this may have forced the islets to aggregate. 4. Prevascularization of empty devices did not produce any significant benefit to inducing normoglycemia. 5. “It is surprising that normal mice had similar fed and post-2-hr glucose challenge values, yet the transplanted mice had fed glucose concentrations that were normal for nude mice but much lower postglucose challenge values.” 69 “Immunodominant Minor ... Indirect Presentation” 1. Study was published in Transplantation in 1998 by Wettstein and team in the United States. 2. Mice spleen cells afforded the cytolytic T lymphocytes (CTLs), and mice were im- planted with the BALB.B cells. 3. Kb and Db molecules present histocompatibility antigens to B6 CTLs, and “B6 anti- BALB.B CTLs recognize a single minor HA peptide that can be eluted from Db molecules [and four Kb-bound peptides].” 4. CTT-1 and CTT-2, the dominant antigens identified in B6 anti-BALB.B CTL response, were alike in their ability to sensitize RMA-S cells for lysis by CTL. 5. “These experiments demonstrated that multiple sets of BALB.B grafts placed on B6 recipients primed for a limited number of dominant antigens detectable by CTLs, but primed for accelerated rejection of multiple allografts that differed from B6 at different, single minor HA that could not be detected with CTLs.” 70 “Function and Survival ... Mice” 1. Study was published in Transplantation in 1998 by Gordon and team in the United States. 2. Mouse islets in planar diffusion chambers, consisting of two membranes and titanium rings, filled with cross linked alginate and barium chloride, were transplanted into diabetes-induced mice. 3. The devices with 250 islets failed to maintain normoglycemia, but those with more islets showed similar effectiveness to each other, providing a ‘lower limit’ of islet number. Two devices performed noticeably better than one. 4. Lower volumes of viable islet issue correlated to hyperglycemia, and greater volumes to normoglycemia. Necrosis of islet volume was drastic directly after implantation due to hypoxia but reduced over time. 5. The surface area required to store islet tissue was impractically high, and new methods must be explored to augment vascularization around implants so packing density can be increased without risking oxygen delivery/cell survival. 71 “Sustained Expression ... Athymic Rodents” 1. Study was published in Human Gene Therapy in 1998 by Johnson and team in the United States. 2. In vivo gene therapy has the risk of the lack of site specificity or controllable expression. Ex vivo has the drawbacks of poor quality control and the possibility of the foreign DNA leading to tumor formation. 3. Human MSU1.2 cells “were transduced with the retroviral vector MFG [...] except that the cDNA for human Factor IX (hF.IX) was encoded,” loaded into TheraCyte capsules, and implanted in athymic mice and rats. 4. In vitro, human Factor IX was detected, but results suggest the cells were not able to fully process it. In vivo, expression steadily increased and went to zero after extraction of the capsules; only transient expression was observed with freely implanted cells. 5. The derivation of an equation, which factors in multiple experimental assumptions, is presented that concludes that approximately 30 devices would need to be implanted in a 70-kg human for treatment of hemophilia. 72 “Use of an Immunoisolation ... Immunotherapy” 1. Study was published in the Annals of the New York Academy of Sciences in 1997 by Brauker and team in the United States. 2. Pore size of encapsulating membranes must be large enough to allow for vascularization, but other factors, such as membrane structure—which can flatten cells or leave them rounded upon entrance to the membrane, reminiscent of foreign body response—affect the process’s success as well. 3. CD4+ T-cells play the dominant role in immune response to implants, as they likely respond to antigens secreted through the membranes by the xenograft; it is also pos- sible that the implanted cells are destroyed through a non-specific immune attack. Restricting pore size to minimize antigen release could lead to malnutrition. 4. Mice implanted with TheraCyte-encapsulated carcinoma cell line, MCA-38, remained tumor-free after introduction of free malignant cells elsewhere in the body. 5. As for the case of existing tumors, encapsulated and free irradiated cells provided the most effective treatment; similar results were found when interleukin-2, a slow- release soluble factor, was encapsulated with the irradiated cells. Thus, direct contact between the immune system and immunizing tumor cells was not necessary to achieve a response. 73 “Immunoisolation ... Presentation of Antigen” 1. Study was published in the Journal of Immunotherapy in 1997 by Brauker and team in the United States. 2. Tumor cells can be put in an immunoisolation device in order to stimulate the immune response against cancer. 3. This method allows for sequestration but extended longevity and eventual removal of the tumor cells. 4. Rodent adenocarcinoma MCA-38 cells were encapsulated and put in mice, who did not develop tumors (unlike the control group) after being injected with free tumors. 5. This success may be owed to antigens in the membranes secreting into the immune system, activating it. 74 “Transplantation of Cells ... Gene Therapy” 1. Study was published in Methods in Molecular Biology in 1997 by Brauker and team in the United States. 2. Factor IX is a protein that aids in coagulation and is deficient in those with hemophilia B. Spontaneous bleeding is a symptom of the condition. 3. “Canine fibroblasts were transduced with a retroviral vector encoding the gene for human Factor IX” and implanted inside a Biopore bilayer membrane using a titanium ring in athymic rats. 4. Although the sera from the rats did not show presence of Factor IX, human Factor IX was found in the encapsulated cells upon explantation. Thus, the release could simply have been too low to be detected by ELISA. 5. Additionally, “primary canine fibroblasts were transduced with a retrovirus encoding canine Factor IX” and implanted, which was detected in rat plasma by ELISA. 75 “Local Inflammatory ... Local Vascularization” 1. Study was published in Transplantation in 1996 by Brauker and team in the United States. 2. Two types of rejection to xenogeneic implants exist: a) hyperacute response, which is immediate and requires vascularization of the implant, and b) cellular rejection, which is more gradual. 3. PTFE/Biopore membranes were used as immunoisolation devices and were loaded with mice or rat embryonic lung tissue and implanted in rats. 4. In comparison to isografts, xenografts had: a) a more severe foreign body response with the presence of lymphocytes, b) low vascularization (although not in athymic subjects), and c) low tissue survival. 5. Thus, xenografts are destroyed by a host immune response whose mechanism is not entirely known, but likely occurs due to secretion of an antigen by the xenogeneic tissue. Pore size must therefore be controlled to reduce rejection. 76 “CD4+ T Cell ... B Cells” 1. Study was published in Transplantation in 1996 by Charlton and team in Australia. 2. Xenografts may in fact induce a milder immune reaction than allografts, after the effects of hyperacute rejection, for “direct T-cell recognition of the allogeneic MHC antigens provokes stronger responses than direct T-cell recognition of xenogeneic MHC antigens.” 3. Monkey kidney cells were loaded into a cell-impermeable Biopore/PTFE/titanium ring membrane and implanted in athymic, immunodeficient, and natural killer (NK) cell- lacking mice, after which monoclonal antibodies were injected into the mice. 4. In immunodeficient mice, xenografts survived and vascularization was found; there was a direct correlation between distance between the membranes and the area of necrosis. Xenografts died in immunocompetent mice and those lacking NK cells. 5. CD4+ T-cells were almost solely responsible for the presence of lymphocytes and macrophages around implants, despite no host-cell contact. Antigens likely perme- ated through the membrane. 77 “Structure and Function ... Nude Mice” 1. Study was published in Hormone and Metabolic Research in 1996 by Hellerstrom and team in Sweden. 2. Human pancreatic islets were macroencapsulated in two membranes—proven to pro- vide immunoisolation and promote neovascularization—supported by titanium rings. 3. The islets implanted in the kidney region of athymic mice and those in the capsules (implanted into the “epididymal fat pad” and demonstrating vascularization) showed similar survival. 4. Non- and encapsulated islets contained nearly equal amounts of insulin, yet rodent islets contained less insulin than their human counterparts. 5. Human C-peptide was found in significant concentrations several weeks after sole im- plantation of the islets. 78 “Neovascularization ... Microarchitecture” 1. Study was published in the Journal of Biomedical Materials Research in 1995 by Johnson and team in the United States. 2. Foreign body response to implanted layers generally takes the form of a sheet of macrophages, multiple fibroblasts layers, and then vascularized tissue. 3. Analysis of FBR on membranes was performed by implantation in rats. 4. Those with large pore sizes showed vascularization at the membrane-tissue interface, so lamination was conducted to reduce pore size. 5. It is thought that “geometry of the membrane influences the behavior of the inflam- matory cells that invade the membrane, causing them to secrete angiogenic factors and/or factors that overcome an existing inhibition of neovascularization;” what is de- sired rather is ‘inertness,’ where neovascularization is developed in a beneficial way for the implant. 79 “Implantable Biohybrid Artificial Organs” 1. Review was published in Cell Transplantation in 1995 by Colton in the United States. 2. The review discusses membrane organ design and how that translated to the function of the encapsulated cells. 3. Oxygen supply of encapsulated cells corresponds to tissue density and the degree of vascularization. 4. The specific processes by which implanted semipermeable membranes are rejected by an immune system are yet to be ascertained. 5. New technologies show great potential for the field of immunoisolation devices. 80 “Allogeneic Ovarian ... Ovariectomized Mice” 1. Study was published in Tissue Engineering by David and team. 2. Women can suffer from reproductive harm by the chemo- and radiotherapy that save them from cancer. 3. Mice ovarian pieces were encapsulated in Poly(ethylene glycol)-Vinyl Sulfone (PEG- VS) hydrogels and TheraCyte capsules and then implanted into adult mice whose ovaries were removed. 4. The Hypothalamus-Pituitary-Gonadal axis was restored in mice who received the im- plant, and allo-antibodies were not found in them (this was not the case for the control group). 5. Immune response was thus not only avoided but endocrine activity was also restored. 81 “CXCL12 Modulation ... Islet Macrocapsulation” 1. Study was published in the American Journal of Transplantation in 2017 by Poznansky and team in the United States. 2. CXCL12-containing Alzet pumps attached to TheraCyte capsules were implanted into mice. 3. They developed diabetes via streptozotocin-induction, and pig islets were transplanted with CXCL12 and sodium alginate into the capsules. 4. Euglycemia was achieved in all mice. 5. CXCL12 contributed to vascularization of capsules and reduced immune response. 82 “Robust, Nanofiber-Enabled ... Delivery” 1. Abstract was presented in the Frontiers in Bioengineering and Biotechnology Confer- ence in 2016 by Ma and team in the United States. 2. Hydrogels such as alginate are an alternative to polymeric phase inverted or expanded Teflon (TheraCyte) encapsulation membranes that are not sufficiently biocompatible. 3. Nanofiberenabled encapsulation devices (NEED’s) were developed by “impregnating the highly porous electrospun nanofiber membranes of pre-made tubular or planar devices with hydrogel precursor solutions and subsequent crosslinking.” 4. PEG and alginate were used for fabrication of the membranes, which demonstrated viability with different compartmentalizations, as well as successful mass transfer and sturdiness. 5. Rat islets implanted in diabetic mice corrected the condition, led to minimal fibrotic overgrowth, and retained live cells. 83 “Progress in Clinical ... Islet Xenotransplantation” 1. Study was published in Transplantation in 2016 by Cozzi and team in the United States, Japan, Argentina, Belgium, Germany, and Italy. 2. A clinical trial involving two low-density encapsulated porcine islet transplants into diabetic humans showed promising results, with few unaware hypoglycemic episodes. 3. “High-mobility group box 1 (HMGB1), a damage associated molecular pattern (DAMP) molecule, is released from transplanted islets and triggers an inflammatory reaction leading to early graft loss of intrahepatic syngeneic islets in mice and autologous islets in humans.” 4. Genetically engineering pigs from which islets will be used can augment insulin produc- tion and better control of glycemia; genetic engineering could also be used to reduce immunosuppressive therapy needed upon transplantation. 5. A discussion regarding ethical/practical considerations of xenotransplantation by the FDA, IXA, WHO, Japan, Switzerland, and more is made. 84 “Nanomaterials and Regenerative Medicine” 1. Book was published in 2016 by Lin and team in Croatia. 2. Stem cells could be very useful in the field of tissue regeneration and engineering. 3. Nanomaterials show great potential and superiority compared to microparticulate mat- ter when it comes to biocompatibility and chemical/physical/magnetic/mechanical properties. 4. The book discusses existing nanotechnologies and their applications to “protective, mechano-sensitive, electro-active, and shear stress-sensitive tissues.” 5. Contributors to the text include researchers from Johns Hopkins University, the Uni- versity of British Columbia, the Oral and Dental Research Institute at the University of the Western Cape in South Africa, and the Nanotechnology Institute in Italy, among others. 85 “Host Response ... Biomaterial Selection” 1. Book was published in 2015 by Badylak and team in the United States. 2. The main goal of the text is to give insight into the ingredients that lead to success of an implant, trends followed by failure, biological interactions, and the future of medical device design and animal trials. 3. Several aspects contributing to host acceptance/rejection of implants such as age, im- mune system, surgical technique, etc. are discussed by their respective experts in the field. 4. Examples of chapters in the book include “The Biocompatibility of Implant Materi- als,” “Role of Dendritic Cells in Response to Biomaterials,” and “Molecular Events at Tissue-Biomaterial Interface.” 5. Contributors to the text include researchers from Stanford University, the McGowan Institute for Regenerative Medicine at the University of Pittsburgh, the Institute of Child Health in the UK, and the Humanitas Clinical and Research Hospital in Italy, among others. 86 “Sustained Subcutaneous ... Myocardial Infarction” 1. Study was published in 2015 in Cardiovascular Research by Lim and team in the United States, Australia, Singapore, Taiwan, and China. 2. TheraCyte capsules were charged with Human W8B2+ cardiac stem cells—or growth media as a control—and implanted in mice, whose left anterior descending coronary arteries were closed off 2 mm below the left atrium to induce heart failure. 3. “Subcutaneous implantation of W8B2+ CSCs encapsulated in the TheraCyte devices can deliver sustained doses of paracrine factors systemically to improve cardiac function and attenuate adverse cardiac remodelling post-MI” (myocardial infarction). 4. Encapsulated cells survived, proliferated, and expressed smooth muscle actin after four weeks, and the capsules vascularized; allogeneic tissue could thus be used in patients without immunosuppression. 5. Extracellular vesicles were more often secreted in TheraCyte capsules than in a 2D monolayer culture, as well as in transduced cells compared to untransduced ones.